Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins

Abstract

Identifying sites of protein-ligand interaction is important for structure-based drug discovery and understanding protein structure-function relationships. Mass spectrometry (MS) has emerged as a useful tool for identifying residues covalently modified by ligands. Current methods use database searches that depend on acquiring interpretable fragmentation spectra (MS2) of peptide-ligand adducts. This is problematic for identifying sites of hydrophobic ligand incorporation in integral membrane proteins (IMPs) where poor aqueous solubility and ionization of peptide-ligand adducts and collision-induced adduct loss hinder the acquisition of quality MS2 spectra. To address these issues, we developed a Fast Ligand Identification (FLI) tag that can be attached to any alkyne-containing ligand via Cu(I)-catalyzed cycloaddition. FLI-tag adds charge to increase solubility and ionization, and utilizes stable isotope labeling for MS1 level identification of hydrophobic peptide-ligand adducts. FLI-tag was coupled to an alkynecontaining neurosteroid photolabeling reagent and used to identify peptide-steroid adducts in MS1 spectra via the stable heavy isotope pair. Peptide-steroid adducts were not identified in MS2-based database searches because collision-induced adduct loss was the dominant feature of CID fragmentation, but targeted analysis of MS1 pairs using electron transfer dissociation (ETD) markedly reduced adduct loss. Using FLI-tag and ETD we identified Glu73 as the site of photo-incorporation of our neurosteroid ligand in the IMP, mVDAC1, and top-down MS confirmed a single site of photolabeling.

Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1

“…Photolabeling efficiency and fragmentation patterns provided by topdown MS complemented our residue level site identification by bottom-up MS. Furthermore, derivatization with FLI-tag and subsequent ETD fragmentation enabled detection of sterolbased peptide adducts and site-defining ions, which are otherwise difficult to identify due to collision cell adduct loss (20).…”

“…These reagents were designed to permit mapping of cholesterol binding pockets by placing the diazirine in different positions. They also contain an alkyne allowing for the attachment of FLItag to facilitate identification of peptide-steroid adducts (20). Top-down MS of photolabeled mVDAC1 was first performed to characterize the efficiency and stoichiometry of labeling.…”

“…Samples were analyzed as previously described (20). Samples were separated on a home packed C18 column (15 cm ϫ 75 M ϫ 200 Å) and introduced to the mass spectrometer with a nanospray source.…”

“…stable isotope-labeled tag, which enables peptide adducts to be identified by an MS1 level search (20). We have also utilized top-down MS to gain complementary information regarding the efficiency, stoichiometry, and sites of photolabeling.…”

Multiple Functional Neurosteroid Binding Sites on GABAAReceptors

“…Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABA A receptors.Electrophysiological studies based on these predictions indicate that both the α 1 intrasubunit and β 3 -α 1 intersubunit sites are critical for neurosteroid action. 5 circumvent challenges associated with mass spectrometric identification (predominantly neutral loss) and quantification of neurosteroid-peptide adducts 35 . Using these approaches we have identified three clusters of neurosteroid photolabeled residues on the human  1  3 GABA A receptor.…”